The present invention relates to novel biologically active composites and uses thereof and, more particularly, to biologically active silver-coated proteins, to integration thereof in biosensors and electronic devices and to pharmaceutical compositions containing and methods of treatment utilizing same. The present invention further relates to processes and intermediates for the preparation of such composites.
Electroless deposition is a widely known technique for depositing metals such as, for example, copper, silver and cobalt, on various surfaces. In principle, electroless deposition is performed in electrolytic solutions or fluids (e.g., aqueous solutions of metal ions) without applying an external voltage, and is effected by an electrochemical reaction between the metal ions and a reducing agent. The electrolytic solution may optionally further include complexing agents and pH adjusting agents and the process can optionally be performed on a catalytic surface (e.g., of a semiconductor wafer).
Electroless deposition is presently known as a highly suitable technique for forming metal films and coatings on microscopic elements and areas on substrates surfaces, for forming barriers and interconnects between different layers on semiconducting wafers and for creating microscopic reservoirs of metallic atoms at specific sites of a subject carrier element.
Hence, at present, electroless deposition is mostly utilized in the manufacture of devices on semiconductor wafers, and particularly in the fabrication of multiple levels of conductive layers, typically of metals such as copper, on a substrate surface.
Electroless deposition is further presently utilized in several biological and medical applications. One example for such an application is the treatment and prevention of tooth cavities, which is effected by depositing a thin metal film onto tooth enamel. The deposited metal films exhibited high adherence to the tooth and maintained the bulk metal properties.
Biosensors such as those disclosed, for example, in U.S. Pat. Nos. 6,773,564, 6,776,888, 6,982,027, 6,984,307, 6,942,770 and Japanese Patent No. 2517153, are analytical devices which convert a biological response into an electrical signal, and thus can quantitatively and qualitatively determine a specific biochemical analyte in a sample. Biosensors can be produced by forming an electrode system having a working electrode (also referred to as “measuring electrode”) and a counter electrode on an electrically insulating substrate, and then forming a reactive layer including, for example, a redox enzyme that reacts with the biochemical analyte. When the reactive layer is in contact with a sample containing the analyte, the analyte is catalytically oxidized by the redox enzyme. The catalytic reaction is typically performed in the presence of an electron-transfer mediator, which is reduced upon the oxidation reaction and is then re-oxidized electrochemically. The concentration of the analyte in the sample is determined upon the recorded oxidation current values.
Another example is the manufacturing of enzyme-containing nanoelectrodes for ultra-sensitive amperometric detection of glucose at low overpotentials. Thus, for example, gold nanotubular electrode ensembles were prepared by electroless depositing the metal within the pores of polycarbonate track-etched membranes. Mono-enzyme glucose oxidase and monolayer/bilayer bi-enzyme glucose oxidase/horseradish peroxidase bioelectrodes were prepared by immobilizing the enzymes onto gold nanotubes surfaces modified with mercaptoethylamine. An advantageous feature of the bi-enzyme electrodes is the possibility to detect glucose at very low applied potentials where the noise level and interferences from other electro-oxidizable compounds are minimal.
Glucose oxidase-containing biosensors for detecting glucose involve a catalytic conversion of glucose to gluconic acid by the enzyme glucose oxidase. This catalytic reaction is coupled to oxygen, and leads to the production of hydrogen peroxide under physiological conditions.
However, electrochemical biosensors based on hydrogen peroxide detection often suffer from substantial inaccuracies, resulting from fluctuations in local oxygen concentrations and the stoichiometric limitation of glucose, known in the field as “oxygen deficit”. This adverse phenomenon may be overcome by introducing synthetic mediators that can react rapidly with the enzyme in its reduced form, and minimize competition with oxygen. Ferrocene (Fc) and derivatives thereof are among the most widely used mediators for that purpose [Dong, S. J. et al., Biosensors & Bioelectronics, 1992, 7, 215-222]. However, the use of Fc and its derivatives as mediators is limited by poor absorption on the electrode surface. Fc and its derivatives are highly soluble in aqueous solutions, especially when in an oxidized/charged form (Fc+), and therefore diffuse away from the immobilized enzyme located on the electrode surface, rendering the whole process inefficient.
Other examples include metallization of various biological moieties by electroless deposition. Thus, electroless deposition of natural arrays of proteins was recently successfully demonstrated for the fabrication of nanowires from microtubules, viral envelopes, amyloid fibers and actin filaments.
The protein metallization described above was effected by techniques that involve nucleation and enlargement by electroless plating. Nucleation was typically performed by adsorption of palladium or platinum ions onto the surface of the biological moiety, followed by chemical reduction thereof, or, alternatively, by surface labeling with colloidal gold particles. Enlargement of the nucleation sites thus obtained into continuously deposited metallic films was typically carried out by immersion in a plating solution containing the metal ions of choice (e.g., Ag+1 or Ni+2) and reducing agents (e.g., NaBH4 or dimethylaminoborane). These techniques typically result in the formation of a relatively thick metal deposition, of e.g., 10 to 35 nanometers. These techniques further lead to the loss of the proteins native biological activity due to deformation and denaturation, blockage of active and binding sites, and gross precipitation of the protein, which most likely results from the strong and incontrollable reducing aptitude of the reducing agent used.
Thus, while the presently known methods for metallizing biological moieties by electroless deposition involve proteins that are either immobilized and/or inactivated before, during and/or as a result of the deposition process, the ability and utility to deposit metals onto a single, soluble biological moiety, particularly protein, while maintaining its activity, dissolvability and other parameters was not demonstrated hitherto. Such a metallization should be performed while maintaining features such as the native chemical structure, the motility and thus the biological activity of the protein. The presently known electroless deposition methods, however, typically interfere with these features and hence do not allow the provision of metallized yet active proteins.
The metallization of proteins while maintaining their activity and/or dissolvability is highly advantageous since it may potentially provide novel therapeutically active agents which may exert, for example, multiples activities resulting from the biologically active protein and/or an active metal coating, and may further be utilized as novel molecular tools for applications such as wiring of nano-sized sensors to composite biochips [I. Wilner and E. Katz. Angew. Chem. Int. Ed. 2000, 39, pp. 1180-1218].
European Patent No. EP00173629B1 teaches the attachment of metal-ion chelating moieties to surface glycans of antibodies, to thereby form conjugates of antibodies and chelating moieties while maintaining the immunoreactivity and immunospecificity of the antibodies. The attachment of the chelating moieties, according to this patent, is effected by generation of aldehyde groups on the surface glycans of the antibody, followed by the conjugation thereto of chelating moieties that have a free amine group, so as to form, under mild conditions, a Schiff-base between the aldehyde group on the antibody's surface and the amine group of the chelating moiety. The resulting conjugate is then used for complexing metal ions via the chelating moieties. This patent, however, fails to teach or suggest the conjugation of reducing moieties that may participate in the more active reduction process involved in electroless deposition, to proteins, while maintaining the activity or dissolvability of proteins, and particularly proteins other than antibodies.